The goals of the work proposed in this supplement to grant No. AM 15119 are to determine in detail the substrate specificities of intestinal peptide hydrolases beginning with the rat brush border enzymes and proceeding to human brush border and cytoplasmic enzymes if time permits, and to determine whether the brush border peptide hydrolases play a significant role in digestion absorption of dipeptides. Substrate specificity of the enzymes isolated will be investigated first by carefully determining the binding affinity of a large number of peptide substrates for individual enzymes. For these studies, synthetic subtrates including Phe-p-nitroanilide and Gly-p-nitroanilide which release the colored compound p-nitroaniline upon hydrolysis will be used for direct spectrophotometric assay of the peptide hydrolases. Binding affinity of large numbers of substrates will be determined by measuring their ability to inhibit hydrolysis of the synthetic substrate. In addition, specific assays will be used to determine the maximum velocity of hydrolysis of selected peptides by each enzyme. Efforts will be made to discern a predictable pattern of substrate hydrolysis on the basis of Km and/or Vmax values. A double labeled dipeptide, C14 Phe-Phe H3, will be used to investigate the rate of absorption of both the C-terminal and N-terminal amino acids by intestinal mucosa. Studies will be performed with flow-through gut sacks, everted rings, and the influx chamber technique. It is reasoned that if the dipeptide is absorbed intact and hydrolyzed inside the cell by cytosol peptide hydrolases, the two labels will show identical absorption. In contrast, if hydrolysis occurs on or in the brush border membrane, it is possible that the two labeled amino acids will be absorbed at different rates due to differences in their binding affinity for the hydrolysis-transport mechanism. Furthermore, any evidence that any compound affects the absorption of one label more than the other will strongly suggest that the peptide is being hydrolyzed on the surface of the cell.